The long term objective of this research is to contribute to our understanding of the basic biochemical and cell biological events involved in the biogenesis of lysosomes. Lysosomes are found in all eucaryotic cells and contain a group of 40-60 different acid hydrolytic (lysosomal) enzymes that is capable of degrading all of the basic biological marcomolecules. The failure to deliver acid hydrolases to lysosomes can result in fatal diseases such as Tay- Sachs and Mucolipidosis II (I cell) disease, therefore it is of both academic and medical interest to understand the mechanisms of lysosome formation. I propose to conduct three lines of investigation on lysosome biogenesis. The first aim is to identify and characterize endogenous membrane markers for the "prelysosomal" endocytic compartment that serves as the delivery site for newly synthesized lysosomal enzymes by preparing monoclonal antibodies (MAbs). One such MAb has tentatively been identified which recognizes a 53 kD membrane protein found predominantly between the putative "prelysosomal" endosomes and secondary lysosomes using biochemical and immunocytochemical methods, 2) to determine the biochemical properties and life histories of the antigens recognized, and 3) to clone and sequence a cDNA for the 53 kD protein. The second aim is to investigate the molecular mechanisms that regulate the delivery of lysosomal enzymes to crinophagic an autophagic vacuoles, tow kinds of secondary lysosomes about which little is known. The third aim is to further characterize mutant Chinese hamster ovary (CHO) cells that are defective in endosomal acidification and the targeting of newly synthesized lysosomal enzymes. So far five complementation groups have been identified, End1-End5. End1 and End2 mutants have been well characterized. Less is known about End3-End5 mutants, so to learn more about these cells, the biosynthesis and post-translational processing of lysosomal enzymes, and the morphological and immunocytochemical events related to lysosome formation will be investigated. The expectation is that all of these studies will significantly add to our understanding of how and where lysosomes are made in mammalian cells.